Matsutake (Tricholoma matsutake) is an important wild edible mushroom with important economic and medicinal value. The sporocarps often occur and develop in shiros. In this study, soils from six shiros of T. matsutake were collected in Yunnan Province and soil dilution plating was used to determine the diversity of bacterial species represented in these samples. Sequencing of 16S rDNA generated 178 sequences from cultured bacteria, representing 38 operational taxonomic units (OTUs) of 4 phyla and 18 genera. The majority of sequences belonged to Proteobacteria (58.43%), Firmicutes (26.40%), Bacteroidetes (10.67%), and Actinobacteria (4.49%). Further analysis revealed that bacteria cultured from these soils were species of Bacillus (Firmicutes, 24.72%), Burkholderia (Proteobacteria, 21.34%), Pseudomonas (Proteobacteria, 11.24%), and Chryseobacterium (Bacteroidetes, 10.67%). Therefore, the diversity of cultured bacteria from the shiros of matsutake in Yunnan Province was abundant.